Monday, February 20, 2012

PCA3 and Prostate Cancer

The FDA has recently approved a PCA3 test assay which is owned by a Canadian company, Gen-Probe. This opens up a whole new avenue for examining PCa amongst men. I examine some of the issue here at a fairly high level.

There has been a great deal of discussion regarding PSA and its lack of sufficient specificity and sensitivity to PCa and there is some evidence that PCA3 will improve the situation. This is yet to be determined in extensive clinical trials. One of the problems with PSA is that it is reflective of total prostate volume and it also naturally increases with age. Thus a male of say 70 years of age and with a 70 cc prostate may easily have a PSA of 2.5 just based upon the size and age factors. 

Likewise if the male were 40 and had a 35 cc prostate then this may be indicative of PCa. In a recent paper by McGarty, we  detailed the issue of PSA sampling and the percent change, ie velocity, as a means to assess the nature of the underlying cause. Namely the more prostate basal cells and luminal cells the higher the PSA. As we shall see there is better correlation with PCA3 but the underlying molecular and cellular dynamics do not appear as well defined at this time, namely we have a marker with no clear underlying genomics cause.

The PCA3 measurement is define as follows:

PCA3 Score = 1000 [mRNA PCA3]/[mRNA PSA]

where [mRNA PCA3] is the concentration of mRNA of PCA3 and the same for the denominator. The range is such that a PCA3 score of less than 5 gives a very low likelihood of PCa and >35 gives a very high probability. The issue here often is repeat biopsy. The suggestion then is that one use PCA3 as a test for repeat biopsy indication (see Gen-Probe PCA3 documentation). Details on ROC for PCA3 are not broadly available and repeatable at this time.

PCA3 was first discussed in 1999 in a paper by Bussemakers et al, at which time it was called DD3. In their abstract the authors stated at the time:

The DD3 gene was mapped to chromosome 9q21–22, and no homology of DD3 to any gene present in the computer databases was found. Our data indicate that DD3 is one of the most prostate cancer-specific genes yet described, and this makes DD3 a promising marker for the early diagnosis of prostate cancer and provides a powerful tool for the development of new treatment strategies for prostate cancer patients.

It further turns out that PCA3 is a noncoding mRNA and thus there is no protein resultant. This was speculated by Bussemakers et al when they published their work in 1999. The key question seems to be why does PCA3 increase when there is a PCa and what is the details of the mechanism. Furthermore where does PCA3 fit within the context of the many pathways we know exist in PCa development.

As Cao an Yao report:

The DD3PCA3 encoding gene is located on chromosome 9 (9q2122). The gene includes four exons and three introns. In PCa, the most frequent mutation is the selective splicing of the second exon. At present, there is a vast body of ongoing studies on PCA3. Hopefully they can further confirm the role of PCA3 in the occurrence and the development of PCa and provide new treatment targets for patients with PCa. Hessels suggested that using quantitative reverse transcriptase polymerase chain reaction (RTPCR) for the detection of urine DD3PCA3 was a valuable molecular detection method in patients with PCa and could help reduce unnecessary biopsies. 

In a multicenter study designed to examine the diagnostic capacity of urine PCA3 detection, the AUC of urine PCA3 detection was 0.66, while the AUC of serum PCA3 detection was merely 0.57. The sensitivity and specificity of PCA3 detection were 65% and 66% , respectively  . Recently, researchers have suggested that serum PSA level plus PCA3 detection was the most promising diagnostic method for PCa . All these studies show that PCA3 is probably an important urine marker for PCa. It also provides a new clue for developing noninvasive detection methods for PCa. Hence, PCA3 may have considerable significance in multiple tumor marker screening of patients for PCa in the future.

Thus one of the questions is what is PCA3 and why does it reflect PCa presence. We know that we are measuring mRNA concentrations, and we know that in measuring them we have experimental evidence that PSA reflects total cell concentration. But what of PCA3, what does that reflect.

In a recent paper by Clarke et al the authors attempt to clarify what the role of PCA3 is.

In order to understand further the importance of the PCA3 gene in PCa we undertook a more detailed investigation of this gene and its chromosomal locus. This investigation points to a considerably more complex transcriptional unit for PCA3 than originally reported including additional novel exons. We describe a number of novel PCA3 splice variants with more specific expression in PCa tissues and metastases. We also demonstrate that PCA3 is embedded in the intron of a second gene, BMCC1, a gene implicated in controlling oncogenic transformation and that both genes showed increased expression in PCa and metastases. The absence of a TATA box element within a human gene promoter has been associated with promiscuous transcriptional initiation. The PCA3 gene does not contain an upstream TATA sequence and it was therefore of interest to determine whether any additional transcription initiation sites existed for PCA3

Perhaps this relationship to BMCC1 may lead to some insight. They continue:

BMCC1 is upregulated in PCa and androgen inducible Since PCA3 is upregulated in PCa and since we showed here that this gene is embedded in a second gene BMCC1, implicated in cellular proliferation, we determined whether BMCC1 was also differentially regulated in PCa. We used a set of RT-PCR primers that span that region of the BMCC1 gene (exons 6 and 7), specific for the full-length BMCC1-1 transcript. Expression of BMCC1-1 was evident in normal prostate and BPH specimens and was upregulated in PCa and metastases. This was confirmed using primers corresponding to the BCH C-terminal region of BMCC1 and for BMCC1-2. 

Indeed amplification of this isoform gave better discrimination between PCa and BPH. Extending these experiments to PCa and other cell lines revealed that both genes were highly expressed, specifically in the PCa cell line LNCaP. In addition BMCC1-1 was detected in a second PCa cell line DU145 but at lower levels. PCA3 is also expressed in DU145 but required further rounds of amplification for detection. The shorter BMCC1 isoforms (BMCC1-3 and/or BMCC1-4) were also detected (using primers specific for the BCH region) in an EBV-transformed lymphoblastoid cell line (JHP), but the longer BMCC1-1 isoform was not detected. Previous data have shown that the level of PCA3 can be induced in LNCaP cells after treatment with dihydrotestosterone, which mimics the effects of binding of the androgen receptor (DHT). We determined whether BMCC1- 1 was also responsive to hormonal induction. The results …. demonstrate that both PCA3 and BMCC1 are maximally induced in the LNCaP cell line at a concentration of 0.5 mM DHT.

Thus there seems to be some means of related induction but again no definitive relationship to well defined pathways.

The following is the PCA3 and PSA ROC for comparison. Note the following (see de la Taille):

The area under the curve of the receiver operating characteristics (AUC ROC) of the PCA3 Score was compared with that of serum total PSA, PSAD and % free PSA. The diagnostic accuracy of the PCA3 Score was statistically significantly better than that of serum total PSA, PSAD and % free PSA. The greatest diagnostic accuracy of the PCA3 Score was obtained at a cut-off of 35: specificity 76% and sensitivity 64% . At a sensitivity of 80%, the PCA3 Score specificity of 58% was higher than the 44% for PSAD and 27% for serum total PSA and % free PSA.

The following from de la Taille is the comparative ROC. It appears that from the limited data available that the ROC curve is substantially better with PCA3 than PSA alone.


The key questions remaining in my mind are:

1. What pathway elements does PCA3 reflect. What genetically is happening and what is the underlying system model. This is always an issue. As with primary medicine we have underlying physiology, here we must have some underlying genomics.

2. What are the cellular mechanism which control PCA3. Again this is a pathways issue.

3. How sustainable is PCA3 ROC for this assay. Many tests have been done and FDA approval is merely acceptance of some limited tests.

4. How does one relate PSA and PCA3. Note that the PCA3 measure does reflect PSA concentration, so we have not abandoned PSA.

5. Why do we normalize PCA3 on PSA? If PSA has such a variability are we normalizing on something which is inherently unpredictable?

References
1.               Bussemakers, M., et al, DD#: A New Prostate specific Gene, Am Assn Cancer Res, 1999.
2.               Clarke, R., New Genomic Structure for Prostate Cancer Specific Gene PCA3 within BMCC1, Plus One March 2008.
3.               de la Taille, A. et al, The PCA3 Assay Improves the Prediction of Initial Biopsy Outcome, 1 CHU Henri Mondor, Paris, France; 2 CHU.
4.               DeMarzo, A., et al, Molecular Alterations in Prostate Cancer as Diagnostic, Prognostic, and Theraputic Targets, Int Soc Uro Path 2008.
5.     McGarty, T.,  PSA Evaluation Methodologies, MIT/RLE Draft Paper 2010, http://www.telmarc.com/Documents/Papers/2010%20PSA%20Evaluation%20Methodologies%20Short.pdf
6.               Rattue, P., Repeat Prostate Biopsies, Medical News Today, February 2012.
7.               Schmidt, U., et al, Quantitative Multi Gene Expression Profiling of Primary Prostate Cancer, The Prostate V 66 pp 1521-1534, 2006.
8.                Wang, R. et al, Rational Approach to Implementation of Prostate Cancer Antigen 3 into Clinical Care, Cancer, Nov 2009.
9.               Wright, J., P. Lange, Newer Potential Biomarkers for Prostate Cancer, Rev Uro V 9 2008 pp 207-213.